Use of adenosine receptor agonists in therapy

ABSTRACT

Use of compounds of formula: (I) wherein R is C 1-4  alkoxy and X is H or OH; for the prevention, treatment, or amelioration of cancer, inflammation, auto-immune disease, ischemia-reperfusion injury, epilepsy, sepsis, septic shock, neurode-generation (including Alzheimer&#39;s Disease), muscle fatigue or muscle cramp is described. The compounds are effective at very low doses, and so can be administered at doses at which serious side effects are not observed.

This invention relates to use of adenosine receptor agonists astherapeutic compounds.

Adenosine is a ubiquitous local hormone/neurotransmitter that acts onfour known receptors, the adenosine A1, A2A, A2B and A3 receptors.Adenosine generally serves to balance the supply and demand of energy intissues. For example, in the heart released adenosine slows the heart byan A1 receptor mediated action in the nodes and atria (Belardinelli, L &Isenberg, G Am. J. Physiol. 224, H734-H737), while simultaneouslydilating the coronary artery to increase energy (i.e. glucose, fat andoxygen) supply (Knabb et al., Circ. Res. (1983) 53, 33-41). Similarly,during inflammation adenosine serves to inhibit inflammatory activity,while in conditions of excessive nerve activity (e.g. epilepsy)adenosine inhibits nerve firing (Klitgaard et al., Eur J. Pharmacol.(1993) 242, 221-228). This system, or a variant on it, is present in alltissues.

Adenosine itself can be used to diagnose and treat supraventriculartachycardia. Adenosine A1 receptor agonists are known to act as powerfulanalgesics (Sawynok, J. Eur J Pharmacol. (1998) 347, 1-11). AdenosineA2A receptor agonists are known to act as anti-inflammatory agents (forexample, from U.S. Pat. No. 5,877,180 and WO 99/34804). In experimentalanimals, A2A receptor agonists have been shown to be effective against awide variety of conditions including sepsis, arthritis, andischaemia/reperfusion injury arising from renal, coronary or cerebralartery occlusion. The common factor in these conditions is a reductionin the inflammatory response caused by the inhibitory effect of thisreceptor on most, if not all, inflammatory cells.

However, the ubiquitous distribution of adenosine receptors means thatadministration of adenosine receptor agonists causes adverse sideeffects. This has generally precluded the development of adenosine-basedtherapies. Selective A1 receptor agonists cause bradycardia. The firstselective A2A receptor agonist(2-[4-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine,or CGS21680), was tested in a Phase 2A clinical trial as a potentialanti-hypertensive. However, administration caused a large fall in bloodpressure and consequent increase in cardiac output. FR 2162128 disclosesthat adenosine derivatives (including 2-alkoxy adenosine derivativescomprising a lower alkyl group of not less than two carbon atoms) havehypotensive and coronary vasodilatory activity.

Bartlett et al (J. Med. Chem. 1981, 24, 947-954) discloses theevaluation of analogues of 1-methylisoguanosine. These analogues include2-methoxyadenosine (also known as spongosine). This and other compoundswere tested for their skeletal muscle-relaxant, hypothermic,cardiovascular and anti-inflammatory effects in rodents following oraladministration (anti-inflammatory activity was assessed by inhibition ofcarageenan-induced oedema in a rat paw). 2-methoxyadenosine caused 25%inhibition of carageenan-induced inflammation in rats at 20 mg/kg po.However, reductions in mean blood pressure (41%), and in heart rate(25%) were also observed after administration of this compound at thisdose.

There is, therefore, a need to provide adenosine receptor agonists thatcan be administered with minimal side effects.

According to the invention there is provided use of a compound of thefollowing formula:

wherein R is C₁₋₄ alkoxy and X is OH;for the manufacture of a medicament for the prevention, treatment, oramelioration of cancer, inflammation, auto-immune disease,ischemia-reperfusion injury, epilepsy, sepsis, septic shock,neurodegeneration (including Alzheimer's Disease), muscle fatigue ormuscle cramp (particularly athletes' cramp).

According to the invention there is also provided use of a compound ofthe following formula:

wherein R is C₁₋₄ alkoxy, and X is H;for the manufacture of a medicament for the prevention, treatment, oramelioration of cancer, inflammation, auto-immune disease,ischemia-reperfusion injury, epilepsy, sepsis, septic shock,neurodegeneration (including Alzheimer's Disease), muscle fatigue ormuscle cramp (particularly athletes' cramp).

In particular, there is provided according to the invention use of acompound of formula I or II for the manufacture of a medicament for theprevention, treatment, or amelioration of inflammatory or auto-immunedisease, including rheumatoid arthritis, osteoarthritis, rheumatoidspondylitis, gouty arthritis, and other arthritic conditions, psoriasis,asthma, chronic obstructive pulmonary disease, fibrosis, multiplesclerosis, endotoxic shock, gram negative shock, toxic shock,hemorrhagic shock, adult respiratory distress syndrome, cerebral malariaTNF-enhanced HIV replication, TNF inhibition of AZT and DDI activity,organ transplant rejection, cachexia secondary to cancer, HIV, chronicpulmonary inflammatory disease, silicosis, pulmonary sarcosis, boneresorption diseases, reperfusion injury (including damage caused toorgans as a consequence of reperfusion following ischaemic episodes e.g.myocardial infarcts, strokes), autoimmune damage (including multiplesclerosis, Guillam Barre Syndrome, myasthenia gravis) graft v. hostrejection, allograft rejections, fever and myalgia due to infection,cachexia secondary to infection or malignancy, cachexia secondary toacquired immune deficiency syndrome (AIDS), AIDS related complex (ARC),keloid formation, scar tissue formation, Crohn's disease, ulcerativecolitis and pyresis, irritable bowel syndrome, osteoporosis, cerebralmalaria, bacterial meningitis, adverse effects from amphotericin Btreatment, adverse effects from interleukin-2 treatment, adverse effectsfrom OKT3 treatment, and adverse effects from GM-CSF treatment.

Compounds of formula (I) or (II) that are selective agonists ofadenosine A2A and/or A3 receptors are particularly preferred because itis believed that such compounds will have strong anti-inflammatoryactivity. By selective agonists of adenosine A2A and/or A3 receptors ismeant agonists that activate adenosine A2A and/or A3 receptors atconcentrations that are lower (preferably one thousandth to one fifth)than required to activate adenosine A1 receptors. Furthermore, A1receptors have pro-inflammatory activity, so such effects are expectedto be minimised for compounds that are selective for A2A and/or A3receptors.

Compounds of formula (I) include: 2-methoxyadenosine, 2-ethoxyadenosine,2-propoxyadenosine, 2-isopropoxyadenosine, and 2-butoxyadenosine.Preferred compounds of formula (I) are 2-methoxyadenosine,2-ethoxyadenosine, and 2-butyloxyadenosine.

Compounds of formula (II) include: 3′-deoxy-2-methoxyadenosine,3′-deoxy-2-ethoxyadenosine, 3′-deoxy-2-propoxyadenosine,3′-deoxy-2-isopropoxyadenosine, and 3′-deoxy-2-butoxyadenosine.Preferred compounds of formula (II) are 3′-deoxy-2-propoxyadenosine,3′-deoxy-2-isopropoxyadenosine, and 3′-deoxy-2-butoxyadenosine.

2-methoxyadenosine has been reported to have an EC50 value at theadenosine A2A receptor of 3 μM (Daly, J. W. et al., (1993) Pharmacol.46, 91-100). However, this compound surprisingly has profoundanti-inflammatory activity at plasma concentrations of 0.2 μM or less.At these low doses 2-methoxyadenosine has reduced probability andseverity of side effects. 2-methoxyadenosine can be administered atconcentrations at which it is effective as an anti-inflammatory, butwhich are below those at which side effects are observed.

Other compounds of formula (I) and compounds of formula (II) are alsobelieved to be much more effective at low doses than other adenosinereceptor agonists. Thus, it is expected that compounds of formula (I)and compounds of formula (II) can be effectively administered at dosesat which they have reduced probability and severity of side effects, orat which side effects are not observed. Such compounds providesignificant advantages over the vast majority of other adenosinereceptor agonists which only have anti-inflammatory effects at the sameconcentrations at which serious side effects are observed.

Compounds of formula (I) or (II) may alternatively or additionally havereduced probability and severity of side effects compared to otheradenosine receptor agonists.

The amount of a compound of formula (I) or (II) that is administered toa subject should be an amount which gives rise to a peak plasmaconcentration that is less than the EC50 value of the compound atadenosine receptors at pH 7.4.

It will be appreciated that the EC50 value of the compound is likely tobe different for different adenosine receptors (i.e. the A1, A2A, A2B,A3 adenosine receptors). The amount of the compound that is to beadministered should be calculated relative to the lowest EC50 value ofthe compound at the different receptors.

Preferably the peak plasma concentration is one thousandth to one fifth,or one fiftieth to one third (more preferably one thousandth to onetwentieth, one hundredth or one fiftieth to one fifth, one fiftieth toone tenth, or one tenth to one fifth) of the EC50 value. Preferably theamount administered gives rise to a plasma concentration that ismaintained for more than one hour between one thousandth and one fifth,more preferably between one thousandth and one twentieth, or onehundredth and one fifth, or one fiftieth and one fifth, of the EC50value of the compound at adenosine receptors at pH 7.4.

For the avoidance of doubt, the EC50 value of a compound is definedherein as the concentration of the compound that provokes a receptorresponse halfway between the baseline receptor response and the maximumreceptor response (as determined, for example, using a dose-responsecurve).

The EC50 value should be determined under standard conditions (balancedsalt solutions buffered to pH 7.4). For EC50 determinations usingisolated membranes, cells and tissues this would be in buffered saltsolution at pH 7.4 (e.g. cell culture medium), for example as in Daly etal., Pharmacol. (1993) 46, 91-100), or preferably Tilburg et al (J. Med.Chem. (2002) 45, 91-100). The EC50 could also be determined in vivo bymeasuring adenosine receptor mediated responses in a normal healthyanimal, or even in a tissue perfused under normal conditions (i.e.oxygenated blood, or oxygenated isotonic media, also buffered at pH 7.4)in a normal healthy animal.

Alternatively, the amount of a compound of formula (I) or (II) that isadministered may be an amount that results in a peak plasmaconcentration that is one thousandth to one twentieth, one thousandth toone third, more preferably one hundredth to one fifth, or one fiftiethto one tenth, of the Kd value at adenosine receptors.

It will be appreciated that the Kd value of the compound is likely to bedifferent for different adenosine receptors (i.e. the A1, A2A, A2B, A3adenosine receptors). The amount of the compound that is to beadministered should be calculated relative to the lowest Kd value of thecompound for the different receptors.

Preferably the amount of the compound that is administered is an amountthat results in a plasma concentration that is maintained for at leastone hour between one thousandth and one fifth, more preferably betweenone thousandth and one twentieth, or one hundredth and one fifth, or onefiftieth and one fifth, of the Kd value of the compound at adenosinereceptors.

The Kd value of the compound at each receptor should be determined understandard conditions using plasma membranes as a source of the adenosinereceptors derived either from tissues or cells endogenously expressingthese receptors or from cells transfected with DNA vectors encoding theadenosine receptor genes. Alternatively whole cell preparations usingcells expressing adenosine receptors can be used. Labelled ligands (e.g.radiolabelled) selective for the different receptors should be used inbuffered (pH7.4) salt solutions (see e.g. Tilburg et al, J. Med. Chem.(2002) 45, 420-429) to determine the binding affinity and thus the Kd ofthe compound at each receptor.

Alternatively, the amount of a compound of formula (I) or (II) that isadministered may be an amount that is one thousandth to one fifth, orone fiftieth to one third (preferably one thousandth to one twentieth,or one hundredth or one fiftieth to one fifth) of the minimum dose ofthe compound that gives rise to bradycardia, hypotension or tachycardiaside effects in animals of the same species as the subject to which thecompound is to be administered. Preferably the amount is one tenth toone fifth of the minimum dose that gives rise to the side effects.Preferably the amount administered gives rise to a plasma concentrationthat is maintained for more than 1 hour between one thousandth and onetwentieth, or one hundredth or one fiftieth and one fifth of the minimumdose that gives rise to the side effects.

Alternatively, the amount of a compound of formula (I) or (II) that isadministered may be an amount that gives rise to plasma concentrationsthat are one thousandth to one fifth, or one fiftieth to one third(preferably one thousandth to one twentieth, or one hundredth or onefiftieth to one fifth) of the minimum plasma concentration of thecompound that cause bradycardia, hypotension or tachycardia side effectsin animals of the same species as the subject to which the compound isto be administered. Preferably the amount gives rise to plasmaconcentrations that are one tenth to one fifth of the minimum plasmaconcentration that causes the side effects. Preferably the amountadministered gives rise to a plasma concentration that is maintained formore than 1 hour between one thousandth and one twentieth, or onehundredth or one fiftieth and one fifth, of the minimum plasmaconcentration that causes the side effects.

It is expected that the amount of a compound of formula (I) or (II) thatis administered should be 0.01 to 15 mg/kg, for example 0.01 to 5 or 10mg/kg. The amount may be less than 6 mg/kg, for example 0.01 to 2 mg/kg.The amount may be at least 0.01 or 0.1 mg/kg, for example 0.1 to 2mg/kg, or 0.2 to 1 mg/kg. A typical amount is 0.2 or 0.6 to 1.2 mg/kg.

Preferred doses for a 70 kg human subject are less than 420 mg,preferably at least 0.7 mg, more preferably at least 3.5 mg, mostpreferably at least 7 mg. More preferably 7 to 70 mg, or 14 to 70 mg.

The dosage amounts specified above are significantly lower (up toapproximately 100 times lower) than would be expected (based on the EC50value of spongosine at the adenosine A2A receptor) to be required forthe compounds of formula (I) to have any beneficial therapeutic effect.

The appropriate dosage of a compound of formula (I) or (II) will varywith the age, sex, weight, and condition of the subject being treated,the potency of the compound, and the route of administration, etc. Theappropriate dosage can readily be determined by one skilled in the art.

Compounds of formula (I) and compounds of formula (II) may beparticularly effective for the prevention, treatment, or amelioration ofparticular types of inflammation, including arthritis (particularly atthe joint capsule of arthritis), asthma, psoriasis, and bowelinflammation.

Compounds of formula (I) and compounds of formula (II) may beparticularly effective in the prevention, treatment, or amelioration ofrheumatoid arthritis, irritable bowel syndrome or osteoarthritis.

There is further provided according to the invention a method ofprevention, treatment, or amelioration of cancer, inflammation,ischemia-reperfusion injury, epilepsy, sepsis, septic shock,neurodegeneration (including Alzheimer's Disease), muscle fatigue ormuscle cramp (particularly athletes' cramp), which comprisesadministering a compound of formula (I) or (II) to a subject in need ofsuch prevention, treatment, or amelioration.

Embodiments of the invention relating to use of a compound of formula(I) (particularly for the prevention, treatment, or amelioration ofinflammation) may exclude 2-methoxyadenosine.

Compounds of formula (I) or (II) may be administered with or withoutother therapeutic agents, for example analgesics (such as opiates,NSAIDs, cannabinoids, tachykinin modulators, or bradykinin modulators)or anti-hyperalgesics (such as gabapentin, pregabalin, cannabinoids,sodium or calcium channel modulators, anti-epileptics oranti-depressants).

In general, a compound of formula (I) or (II) may be administered byknown means, in any suitable formulation, by any suitable route. Acompound of the invention is preferably administered orally,parenterally, sublingually, transdermally, intrathecally, ortransmucosally. Other suitable routes include intravenous,intramuscular, subcutaneous, inhaled, and topical. The amount of drugadministered will typically be higher when administered orally than whenadministered, say, intravenously.

Suitable compositions, for example for oral administration, includesolid unit dose forms, and those containing liquid, e.g. for injection,such as tablets, capsules, vials and ampoules, in which the active agentis formulated, by known means, with a physiologically acceptableexcipient, diluent or carrier. Suitable diluents and carriers are known,and include, for example, lactose and talc, together with appropriatebinding agents etc.

A unit dosage of a compound of the invention typically comprises 5 to500 mg of the active agent. Preferably the active agent is in the formof a pharmaceutical composition comprising the active agent and aphysiologically acceptable carrier, excipient, or diluent. The preferreddosage is 0.1 to 2, e.g. 0.5 to 1, typically about 0.2 or 0.6, mg of theactive agent per kg of the (human) subject. At these levels, effectivetreatment can be achieved substantially without a concomitant fall (forexample, no more than 10%) in blood pressure.

A preferred administration frequency of compounds of the invention isexpected to be two or three times per day.

Compounds of the invention can also serve as a basis for identifyingmore effective drugs, or drugs that have further reduced side effects.

Embodiments of the invention relating to compounds of formula (I) mayexclude 2-propoxyadenosine, and/or 2-isopropoxyadenosine.

Embodiments of the invention relating to compounds of formula (II) mayexclude 3′-deoxy-2-methoxyadenosine and/or 3′-deoxy-2-ethoxyadenosine.

Embodiments of the invention are described in the following exampleswith reference to the accompanying drawings in which:

FIG. 1 shows that 2-methoxyadenosine inhibits carrageenan inducedinflammation without affecting blood pressure;

FIG. 2 shows that 2-methoxyadenosine (0.6 mg/kg p.o.) has no significanteffect on blood pressure or heart rate; and

FIG. 3 shows the change in plasma concentration over time afteradministration of 2-methoxyadenosine.

EXAMPLE 1

FIG. 1: A. 2-methoxyadenosine (62.4 and 624 μg/kg i.p.) inhibitscarrageenan (CGN) induced inflammation with comparable efficacy toindomethacin (3 mg/kg, po), without affecting blood pressure.Carrageenan (2%, 10 microlitres) was administered into the right hindpaw, and the paw volume assessed by plethysomometry. 2-methoxyadenosinewas administered at the same time as carrageenan. 2-methoxyadenosine wasas effective as indomethacin (Indo, 3 mg/kg p.o.).

EXAMPLE 2

FIG. 2: An implantable radiotelemetry device was placed in the abdominalcavity of 6 rats per group. The pressure catheter of the device wasinserted in the abdominal aorta and two electrodes tunnelised under theskin in a lead II position (left side of abdominal cavity/rightshoulder). Individual rats were placed in their own cage on aradioreceptor (DSI) for data acquisition. The effect of 0.6 mg/kg2-methoxyadenosine or vehicle (p.o.) on blood pressure was thenassessed. A: blood pressure; B: heart rate.

EXAMPLE 3

The EC50 value of 2-methoxyadenosine at the adenosine A2A receptor is900 ng/ml (3 μM). FIG. 3 shows the change in plasma concentration overtime after administration of 2-methoxyadenosine at 0.6 mg/kg to a rat.It can be seen that the plasma concentration remains above 2% of theEC50 value for more than 3 hours. Anti-inflammatory effects have beenobserved (without blood pressure changes) when the peak and maintainedplasma concentrations are as low 8 ng/ml (i.e. 2% of the EC50 valuedetermined in vitro). If the peak plasma concentration reaches the 900ng/ml level (i.e. the EC50 value) profound reductions in blood pressureoccur that last for many hours.

1-11. (canceled)
 12. A pharmaceutical composition in unit dose formcomprising up to 500 mg of 2-methoxyadenosine and a physiologicallyacceptable carrier, excipient, or diluent.
 13. A method of prevention,treatment, or amelioration of cancer, inflammation, auto-immune disease,ischemia-reperfusion injury, epilepsy, sepsis, septic shock,neurodegeneration (including Alzheimer's Disease), muscle fatigue ormuscle cramp, which comprises administering a compound of formula (I):

wherein R is C₁₋₄ alkoxy, and X is H or OH; to a subject in need of suchprevention, treatment, or amelioration at a dosage which gives rise to apeak plasma concentration in the subject that is less than the EC50value of the compound at adenosine receptors at pH 7.4.
 14. A methodaccording to claim 13 for preventing, treating, or amelioratingarthritis, bowel inflammation, rheumatoid arthritis, osteoarthritis,rheumatoid spondylitis, gouty arthritis, and other arthritic conditions,psoriasis, asthma, chronic obstructive pulmonary disease, fibrosis,multiple sclerosis, endotoxic shock, gram negative shock, toxic shock,hemorrhagic shock, adult respiratory distress syndrome, cerebral malariaTNF-enhanced HIV replication, TNF inhibition of AZT and DDI activity,organ transplant rejection, cachexia secondary to cancer, HIV, chronicpulmonary inflammatory disease, silicosis, pulmonary sarcosis, boneresorption diseases, reperfusion injury (including damage caused toorgans as a consequence of reperfusion following ischaemic episodes e.g.myocardial infarcts, strokes), autoimmune damage (including multiplesclerosis, Guillam Barre Syndrome, myasthenia gravis) graft v. hostrejection, allograft rejections, fever and myalgia due to infection,cachexia secondary to infection or malignancy, cachexia secondary toacquired immune deficiency syndrome(AIDS), AIDS related complex (ARC),keloid formation, scar tissue formation, Crohn's disease, ulcerativecolitis and pyresis, irritable bowel syndrome, osteoporosis, cerebralmalaria, bacterial meningitis, adverse effects from amphotericin Btreatment, adverse effects from interleukin-2 treatment, adverse effectsfrom OKT3 treatment, and adverse effects from GM-CSF treatment.
 15. Amethod according to claim 13, wherein the compound is administered tothe subject at a dosage that results in plasma concentrations of thecompound in the subject being maintained for more than 1 hour betweenone thousandth and one fifth of the EC50 value of the compound atadenosine receptors at pH 7.4.
 16. A method according to claim 13,wherein the compound is administered to the subject at a dosage thatresults in plasma concentrations of the compound in the subject beingmaintained for more than 1 hour between one thousandth and one fifth ofthe minimum plasma concentration of the compound that gives rise tobradycardia, hypotension or tachycardia side effects in animals of thesame species as the subject to which the compound is to be administered.17. A method according to claim 13, wherein the compound is administeredat a dosage of 0.01 to 15 mg/kg.
 18. A method according to claim 13,wherein the compound is administered at a dosage of 0.1 to 2 mg/kg. 19.A method according to claim 13, wherein the compound is administered ata dosage of 0.6 to 1.2 mg/kg.
 20. Use of a compound of formula (I):

wherein R is C₁₋₄ alkoxy, and X is H or OH; for discovery of drugs forthe prevention, treatment, or amelioration of cancer, inflammation,ischemia-reperfusion injury, epilepsy, sepsis, septic shock,neurodegeneration (including Alzheimer's Disease), muscle fatigue ormuscle cramp.
 21. A method according to claim 13, wherein X is OH.
 22. Amethod according to claim 13, wherein X is H.
 23. A method according toclaim 21, wherein the compound is 2-methoxyadenosine, 2-ethoxyadenosine,or 2-butyloxyadenosine.
 24. A method according to claim 22, wherein thecompound is 3′-deoxy-2-propoxyadenosine, 3′-deoxy-2-isopropoxyadenosine,or 3′-deoxy-2-butoxyadenosine.